EMSA: Electrophoretic Mobility Shift Assay
An EMSA stands for an Electrophoretic Mobility Shift Assay (1). This assay is used to detect protein and nucleic acid interactions. EMSAs are particularly useful when determining areas of DNA that are bound by specific transcription factors. The basic principle is that protein/nucleic acid complexes will migrate at much higher molecular weights than free nucleic acid or protein alone (2). In general, radiolabeled (P-32) DNA is run with cell extracts on a polyacrylamide gel. Once electrophoresis is complete, the gel is examined via autoradiography and bands are observed. If the protein of interest is bound to the DNA, the combination band is said to be "shifted". In addition, antibodies can be used to probe for the specfic protein binding to the DNA. This band conatining the DNA, protein, and bound Ab would be considered a "supershift". Figure 1 shows the expected shifts and supershifts associated with different combinations of probe, protein, antibody and binding competitors. The EMSA was first described by Fried and Collins in 1981 in order to study binding of the lac repressor (lacl) to the lac operon (4). By using this method, the authors were able to determine the binding, site distribution, and kinetics of the repressor protein to the operon DNA site (2, 3). General Protocol for EMSA Protocol adapted from Hellman and Fried, Nature Protocols (2): 1. Prepare a 5-15% polyacrylamide gel depending on the size of the protein/nucleic acid complex expected. 2. Pre-electrophoresis: Loading dye containing glycerol is run at 10V in order to make sure there are no defects in the gel. If there are defects, the protein/DNA band will not run properly and sample will be lost. 3. Sample preparation and electrophoresis: Samples containing DNA only (free probe), protein only, antibody only, DNA+protein, DNA+protein+Ab, Ab+protein, and DNA+antibody are prepared in sample buffer (Laemmli buffer) and incubated together for 30minutes. Titrations of the proper concentrations of DNA and protein will need to be optimized. 4. Electrophoresis: Samples are loaded into the gel and electrophoresis is carried out. 5. Detection of electrophoretic bands using autoradiography: Once gel running is complete, the gel is removed from the electrophoresis apparatus, blot dried, and placed in plastic wrap (no wrinkles or bubbles). In a dark room, X-ray film is taken out, put into a dark cassette, and the gel placed on top of the film. The cassette is then closed and the film is exposed for up to 24hrs at 4C. Film is then developed in an X-ray developer. Figure 2 is a basic outline of the above protocol. Note that each step would require optimization. Uses for EMSA EMSAs have been used in literature to study binding of protein and DNA as there are few alternative assays. Alternatives include footprinting and nitrocellulose filter-binding, with footprinting being a significantly more difficult technique and nitrocellulose filter-binding being less specific than the traditional EMSA. Examples in the literature shows that EMSA is particularly useful in studying the transcription factor NF-kB (nuclear factor kappa light chain enhancer of activated B cells). In the first study, Matsukura et al. evaluated the regulation of eotaxin, a chemokine that recruits eosinophils, by NF-kB and STAT6, two transcription factors upregulated by TNF-a and IL-4 cytokine production. TNF-a and IL-4 are hallmark cytokines produced in patients with asthma. The authors found via EMSA that in response to TNF-a and IL-4, NF-kB and STAT6 respectively bind to the eotaxin promoter, driving transcription of the eotaxin gene and recruitment of eosinophils into the airway (5 ). In the second study by Howard et al., EMSA was performed to demonstrate decreased NFkB activity and increased p53 in patients treated with bortezomib and idarubicin for acute myeloid leukemia. NFkB has been linked to increased AML progression, while p53 is a tumor suppressor protein (6 ). This finding supports the fact that bortezomib and idarubicin can be used in treatment of AML, and that the mechanism of action is to decrease activity of NF-kB. References 1. "Electrophoretic Mobility Shift Assay." Wikipedia. http://en.wikipedia.org/wiki/Electrophoretic_mobility_shift_assay 2. Hellman and Fried. "Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions." Nature Protocols. (2007). 1849-1861. PMID: 17703195 3. Fried and Crothers. "Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis." Nucleic Acids Res. (1981). 9(23):6505-25 PMID: 6275366 4. "Lac Operon, Lac repressor". Wikipedia. 1) http://en.wikipedia.org/wiki/Lac_operon 2) http://en.wikipedia.org/wiki/Lac_repressor 5. Matsukura et al. "Activation of Eotaxin Gene Transcription by NF-kB and STAT6 in Human Airway Epithelial Cells." Journal of Immunology. (1999) 163:(12): 6876-83 PMID: 10586089 6. Howard et al. "A phase 1 study using bortezomib with weekly idarubicin for treatment of elderly patients with acute myeloid leukemia." Leukemia Research. 07 September 2013. S0145-2126 PMID: 24075534